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(Circulation Research. 1999;84:1302-1309.)
© 1999 American Heart Association, Inc.

Original Contributions

Modulation of Iron Uptake in Heart by L-Type Ca²⁺ Channel Modifiers

Possible Implications in Iron Overload

Robert G. Tsushima, Alan D. Wickenden, Ron A. Bouchard, Gavin Y. Oudit, Peter P. Liu, Peter H. Backx

From the Departments of Physiology and Medicine, University of Toronto and The Toronto Hospital, Toronto, Ontario, Canada.

Correspondence to Dr Peter H. Backx, Department of Medicine, The Toronto Hospital, CCRW 3-802, 101 College St, Toronto, Ontario M5G 2C4, Canada. E-mail p.backx{at}utoronto.ca

	Abstract

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Abstract—Heart failure is the leading cause of mortality in patients with transfusional iron (Fe) overload in which myocardial iron uptake ensues via a transferrin-independent process. We examined the ability of L-type Ca²⁺ channel modifiers to alter Fe²⁺ uptake by isolated rat hearts and ventricular myocytes. Perfusion of rat hearts with 100 nmol/L ⁵⁹Fe²⁺ and 5 mmol/L ascorbate resulted in specific ⁵⁹Fe²⁺ uptake of 20.4±1.9 ng of Fe per gram dry wt. Abolishing myocardial electrical excitability with 20 mmol/L KCl reduced specific ⁵⁹Fe²⁺ uptake by 60±7% (P<0.01), which suggested that a component of myocardial Fe²⁺ uptake depends on membrane voltage. Accordingly, ⁵⁹Fe²⁺ uptake was inhibited by 10 µmol/L nifedipine (45±12%, P<0.02) and 100 µmol/L Cd²⁺ (86±3%; P<0.001) while being augmented by 100 nmol/L Bay K 8644 (61±18%, P<0.01) or 100 nmol/L isoproterenol (40±12%, P<0.05). By contrast, uptake of 100 nmol/L ferric iron (⁵⁹Fe³⁺) was significantly lower (1.4±0.3 ng Fe per gram dry wt; P<0.001) compared with divalent iron. These data suggest that a component of Fe²⁺ uptake into heart occurs via the L-type Ca²⁺ channel in myocytes. To investigate this further, the effects of Fe²⁺ on cardiac myocyte L-type Ca²⁺ currents were measured. In the absence of Ca²⁺, noninactivating nitrendipine-sensitive Fe²⁺ currents were recorded with 15 mmol/L [Fe²⁺]_o. Low concentrations of Fe²⁺ enhanced Ca²⁺ current amplitude and slowed inactivation rates, which was consistent with Fe²⁺ entry into the cell, whereas higher Fe²⁺ levels caused dose-dependent decreases in peak current. Fe³⁺ had no effect on current amplitude or decay. Combined, our data suggest that myocardial Fe²⁺ uptake occurs via L-type Ca²⁺ channels and that blockade of these channels might be useful in the treatment of patients with excessive serum iron levels.

Key Words: iron overload • channels • heart failure • permeability • Ca²⁺

	Introduction

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Iron is essential for cellular metabolism and enzyme function. Normally, tissue levels of iron are precisely regulated and stored as a ferritin complex, thereby minimizing the potential toxic effects of catalytically active iron.¹ However, elevations in serum iron occur commonly and are associated with a number of disease conditions. For example, acute iron poisoning is the most common cause of overdose mortality in young children² and often results in myocardial dysfunction.³ On the other hand, chronic elevations of serum iron are associated with a number of disease conditions, including cardiomyopathy, diabetes mellitus, hypopituitarism, and liver cirrhosis.^{4 5 6} Iron overload cardiomyopathy is the most common cause of heart failure in young adults and is often associated with arrhythmias.⁷ It generally results from primary or secondary hemochromatosis.⁷ Primary (or hereditary) hemochromatosis is an autosomal recessive disorder that results from mutations in the histocompatibility antigen HLA-A⁸ and afflicts 10% of people from European extraction.⁹ Secondary hemochromatosis is the most common single gene disorder in humans¹⁰ ; it causes major thalassemic syndromes and sideroblastic anemias associated with ineffective erythropoietic activity and parenchymal iron overload.^{9 11} In addition, iron overload in these patients is often compounded by additional iron loads that result from chronic blood transfusions.¹¹

Currently, no satisfactory therapies exist for the treatment of iron overload disorders. Iron chelators have aided the long-term survival of iron-overload patients¹² and reduce the incidence of cardiac dysfunction.¹³ However, patient compliance is poor with deferoxamine mesylate,¹⁴ and recent evidence suggests that the oral chelator deferiprone is ineffective in thalassemic patients and may promote hepatic fibrosis.¹⁵ Although chelation treatment improves survival,¹² these patients are still at risk for developing late iron-induced cardiomyopathy.⁷ Therefore, understanding the mechanisms involved in iron accumulation in the heart and other tissues may prove useful for the development of new treatment strategies in iron-overload patients.

In most cells, iron uptake is mediated through internalization of the transferrin-iron complex bound to high-affinity membrane receptors.¹⁶ A second mechanism of iron uptake occurs through a transferrin-independent process. This non–transferrin-bound iron (NTBI) transport process is considered to have a minor role in iron uptake under normal physiological conditions but becomes the primary uptake mechanism when serum iron is severely elevated (eg, primary and secondary hemochromatosis). Under these conditions, iron saturation of transferrin and reductions in the number of transferrin receptors occur, which results in excessive transferrin-independent iron uptake via an unknown transporter pathway.^{16 17} NTBI uptake has been demonstrated in a number of mammalian cells, including cardiac myocytes.^{18 19} It is calcium-dependent and can be enhanced by prior iron loading of the cell.^{20 21} Of importance to our studies, it has been shown that a critical step in NTBI uptake is the reduction of ferric iron (Fe³⁺) to the ferrous state (Fe²⁺) by a membrane-associated ferrireductase.²¹

In the present study, we demonstrate that a significant component of myocardial uptake of reduced iron (ie, Fe²⁺) is dependent on the electrical excitability of the heart and can be modulated by agents and interventions that affect the L-type Ca²⁺ channel activity. In addition, we show that Fe²⁺ permeates Ca²⁺ channels at high concentrations and can alter channel kinetics at lower concentrations. Our results suggest that L-type Ca²⁺ channels might contribute significantly to iron uptake by the heart and has many of the properties associated with the unknown NTBI uptake pathway.

	Materials and Methods

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⁵⁹Fe Uptake in Isolated Perfused Hearts
Hearts were rapidly excised from heparinized and anesthetized (sodium pentobarbital, 75 mg/kg) male Sprague-Dawley rats (Charles River, Montreal, Canada; 200–250 g) and mounted for perfusion using the Langendorff technique. Isolated hearts were perfused with Tyrode's solution (8 mL/min) containing (in mmol/L) 140 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 20 butanedione monoxime, 10 glucose, 10 HEPES (pH 7.4 with NaOH), and gassed with 100% O₂. Temperature was maintained at 37°C. The addition of 5 mmol/L ascorbic acid to the perfusion media maintained the iron in the reduced state (Fe²⁺) and prevented the formation of Fe³⁺. For ferric (Fe³⁺) iron experiments, nitriloacetate (5 mmol/L) was used to prevent Fe³⁺ precipitation^{20 21} and the concentration of CaCl₂ was adjusted to maintain a 1 mmol/L free Ca²⁺ level. After a 10-minute equilibration period to remove blood from the vasculature and to ensure proper vascular perfusion, hearts were perfused for 15 minutes with 100 nmol/L ⁵⁹Fe²⁺ (⁵⁹FeSO₄, 27 to 31 mCi/mg; NEN Life Science Products) or 100 nmol/L ⁵⁹Fe³⁺ (⁵⁹FeCl₃, 27 mCi/mg; NEN) then perfused with ice-cold Tyrode's solution for 10 minutes to remove ⁵⁹Fe from the vascular lumen. To reduce nonspecific ⁵⁹Fe²⁺ binding to the perfusion system, 10 µmol/L FeCl₂ or FeCl₃ was included in the perfusion solution. Hearts were blotted dry, and atrial and connective tissues were removed; ⁵⁹Fe²⁺ uptake was determined by gamma counting. Nonspecific ⁵⁹Fe²⁺ uptake was calculated by subtracting the radioactivity measured in the presence of 1 mmol/L FeCl₂. Electrical activity was arrested in hearts with 20 mmol/L KCl. Nifedipine (10 µmol/L; Sigma Chemical Co), (-)Bay K 8644 (100 nmol/L; Research Biochemicals International), or isoproterenol (100 nmol/L; Sigma) was added to the perfusion solution. Stock nifedipine (10 mmol/L) and (-)Bay K 8644 (1 mmol/L) were dissolved in ethanol. The final concentration of ethanol (0.1%) had no effect on ⁵⁹Fe²⁺ uptake.

Electrophysiology
Whole-cell Ca²⁺ currents were recorded at room temperature from enzymatically isolated rat cardiac ventricular myocytes²² using the patch-clamp technique²³ (Axopatch 200A, Axon Instruments). L-type Ca²⁺ currents were elicited by 200-millisecond step depolarizations between –-40 and 70 mV from a holding potential of –-80 mV. A 100-millisecond prepulse to –-45 mV was used to inactivate Na⁺ channels. Current records were sampled at 150 µs and filtered at 2 kHz (4-pole Bessel filter, -3 dB). The external solution contained (in mmol/L) 140 N-methyl-D-glucamine, 2 CaCl₂, 1 MgSO₄, 3 4-aminopyridine, 10 glucose, and 10 HEPES (pH 7.4 with methanesulfonic acid). Ascorbic acid (5 mmol/L) or NTA (5 mmol/L) was included in the external solution for the ferrous and ferric iron experiments, respectively. With NTA in the external solution, the levels of CaCl₂ were adjusted to maintain a free concentration of 2 mmol/L. The pipette solution consisted of (in mmol/L) 140 N-methyl-D-glucamine, 5 MgATP, 2 phosphocreatine, 0.2 GTP, 5 BAPTA, and 10 HEPES (pH 7.2 with methanesulfonic acid). At the end of the experiments, 20 µmol/L nitrendipine was used to determine dihydropyridine-sensitive currents. Nitrendipine was used for these experiments instead of nifedipine to minimize photoinactivation.²⁴

Data Analysis
Data acquisition and analysis was performed using custom written and Origin software (MicroCal, Inc). The dose-response relationship was fit with a Hill equation: I_B/I_O=1/(1+[Fe²⁺]ⁿ/IC₅₀ⁿ), where IC₅₀ is the half-maximal inhibitory concentration of Fe²⁺ and n is the Hill coefficient. Data are presented as the mean±SEM. Statistical analysis was performed by use of a 1-way ANOVA followed by a multiple comparison testing (Student-Newman-Keuls; (SPSS 7.5; SPSS). A P value <0.05 was used to denote statistical differences between groups.

	Results

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Iron Uptake in Isolated, Perfused Hearts
Iron uptake in most mammalian cells is mediated primarily through a transferrin-dependent process, but under conditions of iron overload, NTBI uptake becomes the predominant mode of iron uptake.^{18 20} In rat myocardium, NTBI uptake can exceed the transferrin-dependent pathways by 300-fold.¹⁸ Because the redox state of iron is critical in NTBI uptake and Fe²⁺ is the primary ionic species translocated across the cell membrane,^{21 25} we initially examined iron uptake into myocardium by perfusing isolated rat hearts (Figure 1) with 100 nmol/L ⁵⁹Fe²⁺. This concentration is well below the plasma levels of NTBI measured in patients with hemochromatosis (ie, 1 to 20 µmol/L; References 26²⁶ –28) or in some children after acute iron poisoning (ie, >1 mmol/L; Reference 29²⁹ ). Perfusion of rat hearts for 15 minutes resulted in specific ⁵⁹Fe²⁺ uptake of 20.4±1.9 ng Fe per g of dry wt (n=7). Electrically arresting the hearts with 20 mmol/L KCl reduced ⁵⁹Fe²⁺ uptake to 8.1±1.3 ng Fe per g dry wt (n=8; P<0.01; Figure 1), demonstrating that a major component of myocardial Fe²⁺ uptake is voltage-dependent.

View larger version (30K): [in this window] [in a new window]   Figure 1. Specific 59Fe2+ uptake of isolated perfused rat hearts is voltage-dependent. Hearts were perfused with 59Fe2+ (100 nmol/L) for 15 minutes followed by a 10-minute washout period in the absence and presence of KCl (20 mmol/L), nifedipine (1 or 10 µmol/L), Cd2+ (100 µmol/L), Bay K 8644 (100 nmol/L), or isoproterenol (100 nmol/L). 59Fe uptake was measured from ventricular tissue and normalized to dry wt. Data represent the mean±SEM of 6 to 8 hearts. *P< 0.05, **P< 0.01.

Under normal physiological conditions, voltage-gated cardiac L-type Ca²⁺ channels are selectively permeable for Ca²⁺ versus Na⁺ and K⁺.^{30 31} However, these channels are permeable to other divalent cations, such as Ba²⁺, Sr²⁺, Mn²⁺, and Zn²⁺.^{32 33 34} Therefore, we hypothesized that L-type Ca²⁺ channels contribute to ⁵⁹Fe²⁺ uptake. Consistent with our expectations, 10 µmol/L nifedipine, an L-type Ca²⁺ channel antagonist,³⁵ suppressed myocardial contractility in the isolated perfused rat hearts and decreased ⁵⁹Fe²⁺ uptake to 11.2±2.5 ng Fe per g dry wt (n=6, P<0.02, Figure 1), a level not significantly different from KCl-arrested hearts (P>0.26). A lower concentration of nifedipine (1 µmol/L) produced a more modest reduction in ⁵⁹Fe²⁺ uptake (17.9±0.7 ng Fe per g dry wt, n=6). Conversely, augmenting Ca²⁺ channel activity with the specific L-type Ca²⁺ channel agonist^{35 36} (-)Bay K 8644 significantly increased ⁵⁹Fe²⁺ uptake to 32.9±3.7 ng Fe per g dry wt (n=7; P<0.01), which is 2.3-fold greater than the nifedipine-sensitive component. Because L-type Ca²⁺ channel activity is enhanced by ß-adrenergic receptor activation,^{37 38} 100 nmol/L isoproterenol was added to the perfusion media. This agent caused a significant 40% enhancement of ⁵⁹Fe²⁺ uptake (28.6±2.5 ng Fe per g of wt; n=6, P<0.05). Next, we examined the effects of the inorganic divalent cation Cd²⁺, which blocks L-type Ca²⁺ channels.^{31 34} As shown in Figure 1, 100 µmol/L Cd²⁺ markedly inhibited ⁵⁹Fe²⁺ uptake by 86% (2.9±0.6 ng of Fe per g dry wt, n=6; P<0.001). The inhibition of ⁵⁹Fe²⁺ uptake by Cd²⁺ was significantly greater (P<0.01) than with KCl or nifedipine, suggesting that transporters other than L-type Ca²⁺ channels may be involved in myocardial iron uptake. This is not unexpected because Cd²⁺ interferes with numerous other membrane transporters including iron transporters.^{21 39}

In contrast to these findings, uptake of radioactive oxidized iron (⁵⁹Fe³⁺) by the perfused rat hearts (1.4±0.3 ng Fe per g dry wt, n=4) was 15-fold less than ⁵⁹Fe²⁺ uptake (P<0.001). These results are consistent with previous publications showing that ferrous iron (Fe²⁺) is the primary species entering the heart via the NTBI mechanism.^{21 25}

Fe²⁺ Permeation of L-Type Ca²⁺ Channels
The dependence of ⁵⁹Fe²⁺ uptake on cellular excitability and agents that affect L-type Ca²⁺ channel function led us to examine the interaction of Fe²⁺ with the cardiac L-type Ca²⁺ channel. With 2 mmol/L Ca²⁺ in the external solution, Ca²⁺ currents (I_Ca) peaked at 0 mV (-7.8±0.9 pA/pF, n=8; Figure 2A and 2E). Replacement of external Ca²⁺ with 15 mmol/L Fe²⁺ reduced, but did not eliminate, the amplitude of the inward current (-0.20±0.03 pA/pF at 20 mV; n=8; Figure 2B and 2E). Subsequent exposure of the cells to 20 µmol/L nitrendipine completely blocked all inward current (Figure 2C). The nitrendipine-sensitive Fe²⁺ currents showed little current inactivation (Figure 2D). In addition, activation of the nitrendipine-sensitive Fe²⁺ currents was shifted by 16±2 mV (n=8), resulting in a large rightward shift in the peak of the current-voltage relationship with no measurable change in the reversal potential (Figure 2E). The depolarizing shift in the voltage dependence of activation most probably resulted from the screening of negative surface charges caused by the higher concentration of divalent cation concentration in the external media (15 mmol/L Fe²⁺ versus 2 mmol/L Ca²⁺)⁴⁰ as observed at high external Ca²⁺ and Ba²⁺ concentrations.⁴¹ These results demonstrate that Fe²⁺, like other divalent cations, is capable of permeating the L-type Ca²⁺ channel and shifting gating properties of L-type Ca²⁺ channels.⁴²

View larger version (18K): [in this window] [in a new window]   Figure 2. Nitrendipine-sensitive Fe2+ currents in the absence of Ca2+. A, Whole-cell ICa recorded from rat ventricular myocytes in the presence of 2 mmol/L Ca2+ in the external bath solution. Holding potential was -80 mV, and the test potentials were from -40 to -10 mV. B, Currents recorded after replacement of Ca2+ with 15 mmol/L Fe2+ followed by the application of 20 µmol/L nitrendipine (C). Test potentials ranged from -30 to 0 mV. D, Nitrendipine-sensitive Fe2+ currents measured by the different currents in the absence (B) and presence (C) of the dihydropyridine antagonist. E, Current-voltage relationship of Ca2+ (•) and Fe2+ currents ().

Under more physiological conditions, Fe²⁺ might compete with Ca²⁺ within the permeation pathway for ion conduction, as has been observed with Ba²⁺, Na⁺, and other cations.^{43 44} Therefore, the interaction of Fe²⁺ on I_Ca with 2 mmol/L Ca²⁺ present in the extracellular solution was examined. There was no significant change in peak I_Ca with 250 µmol/L Fe²⁺ in the external solution (2±2%, n=5); however, 500 µmol/L Fe²⁺ potentiated I_Ca by 21±3% (n=5, P<0.01; Figure 3A and 3C). At higher concentrations, Fe²⁺ reduced the peak I_Ca in a dose-dependent manner, with an IC₅₀ of 2.1 mmol/L (Figure 3A and 3C), which is remarkably similar to previous estimates of 2.7 mmol/L, determined in single-channel studies.³⁰

View larger version (14K): [in this window] [in a new window]   Figure 3. Fe2+ potentiates and blocks L-type Ca2+ currents. A, Whole-cell L-type Ca2+ currents from -40 to 0 mV in 10 mV increments evoked from a holding potential of -80 mV in the (a) absence, and presence of (b) 0.5, (c) 2, and (d) 4 mmol/L Fe2+. External Ca2+ was maintained at 2 mmol/L. B, Fe2+ slows ICa inactivation. Whole-cell currents normalized to peak current in the (a) absence and presence of (b) 0.5, (c) 2 and (d) 4 mmol/L Fe2+. The Fe2+-induced slowing in ICa decay was concentration-dependent. C, Concentration-dependent effect of Fe2+ on peak ICa. Peak ICa in the presence of Fe2+ (IB) is expressed as a fraction of peak ICa in the absence of Fe2+ (IO). The line is the best fit of the data to the Hill equation (see Materials and Methods). The data represent the mean±SEM of 5 to 8 cells.

Concomitant with the reduction in peak I_Ca, current decay was slowed in a dose-dependent manner (Figure 3B). To quantify the effects of Fe²⁺ on current inactivation, the fraction of the current at the end of the 200-millisecond depolarization to the peak current at 0 mV (r₂₀₀) was plotted as a function of the Fe²⁺ concentration (Figure 4A).^{45 46} In the absence of Fe²⁺, r₂₀₀ was 0.11±0.02 (n=8), similar to previously reported values.⁴⁵ In the presence of 500 µmol/L Fe²⁺, r₂₀₀ significantly increased to 0.20±0.02 (n=5; P< 0.05) and to 0.48±0.04 at 4 mmol/L Fe²⁺ (n=5; P<0.01). These alterations in inactivation kinetics might affect the net influx of Ca²⁺ into the cell and intracellular Ca²⁺ concentrations despite reductions in the peak current. Indeed, the time integral of the current traces in Figure 4B reveal that the total charge influx increased from 0.38±0.03 pC/pF in control cells to 0.59±0.07 pC/pF (P<0.01) in the presence of 500 µmol/L Fe²⁺. The time integral of the currents gradually decreased at Fe²⁺ concentrations >2 mmol/L (Figure 4B). Thus, Fe²⁺ slows the decay of the Ca²⁺ current in a dose-dependent manner, and at concentrations <500 µmol/L, it increases Ca²⁺ influx through the channel.

View larger version (34K): [in this window] [in a new window]   Figure 4. A, Fractional ICa at 200 milliseconds (r200). The ratio of ICa at the end of the 200 millisecond depolarizing test pulse relative to the peak current at 0 mV in the absence and presence of Fe2+. B, Time integral of ICa at 0 mV in the absence and presence of Fe2+. External solution contained 2 mmol/L Ca2+. Data are the mean±SEM of 5 to 8 cells. *P<0.05, **P<0.01.

The effects of iron on the L-type Ca²⁺ channel were specific for the reduced form of iron (ie, Fe²⁺). Application of ferric iron (2 mmol/L Fe³⁺) in the presence of 2 mmol/L external Ca²⁺ (Figure 5A) did not affect peak current as observed by the current-voltage relationship shown in Figure 5B and more importantly did not affect current decay (Figure 5A). These data suggest that uptake of myocardial iron via the L-type Ca²⁺ channel is mediated by Fe²⁺, not Fe³⁺, permeation.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of Fe3+ on L-type Ca2+ currents. A, Whole-cell ICa recorded in the absence and presence of 2 mmol/L Fe3+ (FeCl3). Currents traces were elicited by a 200-millisecond step depolarization to 0 mV from a holding potential of -80 mV. B, Current-voltage relationship of ICa before and after application of 2 mmol/L Fe3+. Data represent the mean±SEM of 4 cells.

The modulation of L-type Ca²⁺ current by Fe²⁺ may result from nonspecific modification of the channel protein such as sulfhydryl oxidation due to Fe²⁺-dependent free radical production.¹ However, the effects on both peak current and inactivation were reversible when the Fe²⁺ concentration was changed from 500 µmol/L to 2 mmol/L and then back to 500 µmol/L (Figure 6). These results suggest that irreversible modification of the channel protein did not occur and therefore is not responsible for the observed effects on channel inactivation.

View larger version (31K): [in this window] [in a new window]   Figure 6. Reversible effects of Fe2+ on peak ICa and current decay. A, Whole-cell ICa recorded under control conditions (a) followed by exposure to 0.5 (b) and 2 mmol/L Fe2+ (c) and by a second exposure to 0.5 mmol/L Fe2+ (d). Currents were elicited from -40 mV to 0 mV at a holding potential of -80 mV. B, Current-voltage relationship of ICa in the absence (•) and presence of 0.5 (, ), 2 (), and 4 mmol/L Fe 2+ ().

	Discussion

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The present study demonstrates that myocardium Fe²⁺ uptake is inhibited in electrically arrested nonbeating hearts and is modulated by agents that affect L-type Ca²⁺ channel activity. Iron uptake was inhibited by 50% in arrested and nifedipine-treated hearts, consistent with the hypothesis that Fe²⁺ uptake into heart can occur via L-type Ca²⁺ channels. However, in addition to blocking L-type Ca²⁺ channels, the high concentrations of nifedipine used in the studies (Figure 1) can also inhibit other ion channels^{47 48 49} that might contribute to ⁵⁹Fe²⁺ uptake, although no previous studies have demonstrated that Fe²⁺ permeation occurs through these channels. On the other hand, the selective L-type Ca²⁺ channel agonist Bay K 8644 produced a 2.3-fold increase in the nifedipine-sensitive Fe²⁺ uptake by the heart. Assuming complete block of L-type Ca²⁺ current by nifedipine (see below), this Bay K 8644–dependent enhancement of Fe²⁺ uptake matches the 1.5- to 2-fold increase of current produced by these agents in previous voltage-clamp studies.³⁵ By contrast, minimal uptake of ⁵⁹Fe³⁺, which does not permeate L-type Ca²⁺ channels, occurred in these experiments. Overall, these studies suggest that NTBI (ie, Fe²⁺) uptake into the heart can occur, although probably not exclusively (see below), via L-type Ca²⁺ channels.

Under our experimental conditions, the extent of Fe²⁺ blockade by Cd²⁺ was 2-fold greater than by 10 µmol/L nifedipine. The differences between these 2 blockers of L-type Ca²⁺ channels in our experiments may result at least in part from incomplete blockage of Ca²⁺ channels by nifedipine, because nifedipine binding to L-type channels is very voltage-dependent.^{35 50} On the other hand, Cd²⁺ can inhibit many membrane transporters in addition to Ca²⁺ channels; therefore, it seems likely that a second nifedipine-insensitive iron uptake process exists in the myocardium. One potential candidate transporter is the voltage-dependent divalent-cation transporter (DCT1), which was cloned from rat duodenum and appears to be expressed in heart.⁵¹ However, Cd²⁺ is actually transported by DCT1,⁵¹ which would not readily explain our observed effects of Cd²⁺ on Fe²⁺ uptake. Although both NTBI uptake observed in the present study and DCT1 Fe²⁺ uptake display similar voltage dependency, it is unknown whether high K⁺, nifedipine, Bay K 8644, or phosphorylation affect DCT1 transport properties. In contrast to DCT1, voltage-independent Fe²⁺ uptake has been measured in cultured neonatal rat cardiac myocytes.²⁵ In these studies, 20 µmol/L nifedipine inhibited 20% of ⁴⁵Ca²⁺ uptake in cultured myocytes.²⁵ This modest effect of nifedipine is not entirely inconsistent with our results because previous studies have demonstrated that L-type Ca²⁺ channel densities and activity are much lower in cultured neonatal myocytes than adult heart cells.⁵² Clearly, additional studies are required to determine whether multiple pathways for NTBI (ie, Fe²⁺) uptake exist in heart and to characterize their relative importance in iron loading.

Our ability to measure Fe²⁺ currents in the absence of external Ca²⁺ and the effects of Fe²⁺ on inactivation kinetics of L-type Ca²⁺ currents further support the hypothesis that Fe²⁺ permeates the L-type Ca²⁺ channel. This conclusion is consistent with previous studies demonstrating that Fe²⁺ and other transition metals (Zn²⁺, Co²⁺, and Mn²⁺) can permeate L-type Ca²⁺ channels in addition to impeding Ca²⁺ flux.^{30 31 33} However, permeation of divalents like Fe²⁺ through L-type Ca²⁺ channels is too slow to be readily detected using electrical recordings^{30 34} under relevant pathophysiological conditions. Previously, it has been suggested that measurement of intracellular accumulation would provide better evidence for the flux of divalent cations, like Fe²⁺, through L-type Ca²⁺ channels.³⁴ Initially, we attempted to use an optical fluorescence method in isolated cardiac trabeculae and single myocytes. However, these methods were also relatively insensitive partly because redox cycling of iron occurs after Fe²⁺ enters myocytes (see below), as demonstrated previously by the measurable Fe³⁺ labile pools in iron-loaded cardiac myocytes.¹⁹ Our radioisotope ⁵⁹Fe²⁺ flux measurements in the whole hearts avoided these problems, thereby allowing estimation of the rate of iron accumulation in heart.

The nifedipine-sensitive component of iron uptake in our Langendorff experiments is remarkably similar to the 14 to 19 pmol · min^-1 · g^-1 dry wt of Fe²⁺ that is predicted from our electrophysiological studies and those of others³⁰ to enter myocytes via L-type Ca²⁺ channels. This calculation, outlined below, requires that the relative flux of Fe²⁺ versus Ca²⁺ is proportional to the ratio of their maximum current densities (or binding rates) and that binding isotherms adequately describe the dependence of current on the permeant divalent ion concentrations as established previously.⁵³ Since the Fe²⁺ current density in rat ventricular myocytes at 0 mV was 0.20 pA/pF with 15 mmol/L Fe²⁺ and the dissociation constant was 2.1 mmol/L (similar to the 2.7 mmol/L reported previously³⁰ ), the maximum current (I_MAX) for Fe²⁺ is estimated to be 0.23 pA/pF. The corresponding I_MAX for Ca²⁺ current density at 0 mV was 38 pA/pF using an estimated IC₅₀=14 mmol/L.⁵³ Thus, the ratio of maximum currents is estimated to be 6.7x10^-3, which matches closely the corresponding ratio of 7.6x10^-3 that is based on the second order binding rate constants (ie, 3.4x10⁶ mol · L^-1 · s^-1 for Fe²⁺ versus 4.5x10⁸ mol · L^-1 · s^-1 for Ca²⁺).³⁰ The net Ca²⁺ flux that enters a typical myocyte in each beat at 2 mmol/L [Ca²⁺]_o is 16 pC.⁵⁴ Therefore, the predicted maximum net Ca²⁺ flux is 66.4 µmol Ca²⁺ per minute per gram of dry weight, assuming there are 1x10⁸ myocytes per heart, the dry wt–wet wt ratio is 0.2, and the heart rate is about 200 bpm. In contrast, the predicted flux of Fe²⁺ at a concentration of 100 nmol/L is 19.1 pmol · min^-1 · g^-1 dry wt. Corresponding estimates with the ratios of the second-order rate constants to estimate the Fe²⁺ flux predict an uptake rate through the L-type Ca²⁺ channels of 14.3 pmol · min^-1 · g^-1 dry wt.

The above estimates suggest that sufficient Fe²⁺ can enter through L-type Ca²⁺ channels to account for the nifedipine-sensitive Fe²⁺ accumulation observed in our whole heart experiments, provided that extrusion is relatively slow. Is this, in fact, likely to be the case? Previous studies have established that in conditions of iron overload, NTBI entry into cells bypasses the transferrin-based system and overwhelms the normal regulatory capacity of the cell for iron.^{7 16} Consequently, NTBI entering the cell is not bound to ferritin but becomes weakly bound as low-molecular-weight complexes and subsequently undergoes redox cycling of iron.⁵⁵ This not only produces free radicals but also leads to the irreversible precipitation of iron in the form of hemosiderin.⁵⁵ Indeed, it is known that very little, if any, of the NTBI that accumulates in cardiac myocytes under conditions similar to those in our experiments is transported back out of the cell unless the cell is treated with iron chelators,^{19 56} suggesting that Fe²⁺ that enters the cell under these conditions is effectively trapped.

If L-type Ca²⁺ channels contribute to iron uptake in heart, it is reasonable to ask whether this uptake pathway can account for the iron levels observed clinically. Patients with secondary hemochromatosis often have total serum iron levels of 20 to 61 µmol/L, with estimated NTBI of 1 to 20 µmol/L.^{26 27 28} Under these conditions, the amount of iron accumulation predicted to occur via the L-type Ca²⁺ channels in 10 to 15 years, a relevant period for patients not receiving chelation therapy,¹⁴ would be 3 to 5 mg of iron per gram of heart. This compares favorably with the 2 to 8 mg of iron per gram of heart typically observed in these patients.^{19 57}

The Fe²⁺-mediated slowing of Ca²⁺ current inactivation in our studies is analogous to the slowing of Ca²⁺ current inactivation by Ba²⁺ after Ca²⁺ permeates the channel pore.^{45 46 58} Accordingly, this slowing could arise from competition between Fe²⁺ and Ca²⁺ for the C-terminal cytoplasmic Ca²⁺ binding site involved in Ca²⁺-mediated inactivation of L-type Ca²⁺ channels.^{45 46} It is, nevertheless, also conceivable that these effects could result from Fe²⁺ uptake via an independent yet unidentified transporter. But this seems somewhat unlikely because previous single-channel studies have established that the transporter would need to be localized in very close proximity to the Ca²⁺ channel⁵⁸ in order to explain our observations. Alternatively, slowed inactivation by Fe²⁺ might also be due to irreversible oxidation of the channel as a result of free radical production or sulfhydryl oxidation as reported previously.^{59 60} However, the observed effects of Fe²⁺ on current inactivation rapidly reversed following washout, which is inconsistent with an oxidation-based mechanism because reversal requires the application of free radical scavengers or sulfhydryl reducing agents.^{59 60}

The Fe²⁺-induced slowing of Ca²⁺ current inactivation could have a number of important consequences. For example, the slowing of current decay by 500 µmol/L Fe²⁺ resulted in a 50% increase in the time integral of the Ca²⁺ current and thus net Ca²⁺ influx. This is expected to significantly increase intracellular Ca²⁺ levels⁶¹ and possibly contribute to contractile dysfunction (Ca²⁺ overload) or impaired diastolic function observed during the early stages of iron overload.⁷ On the other hand, slowed inactivation of L-type Ca²⁺ currents would increase NTBI Fe²⁺ entry into the myocytes, which may explain the upregulation of Fe²⁺ uptake that was previously reported to occur in iron-loaded cardiac myocytes.^{19 25}

In summary, our data supports the hypothesis that NTBI uptake and iron accumulation by myocardium occurs via L-type Ca²⁺ channels. Consistent with this assertion, we have recently observed a 2-fold reduction in myocardial iron content and mortality with L-type Ca²⁺ channel blockers amlodipine and verapamil using an in vivo murine model of cardiac iron overload (G.Y.O. and P.H.B., unpublished data, 1998). Poor patient compliance with deferoxamine treatment¹⁴ and the lack of efficacy with the oral iron chelator, deferiprone,¹⁵ warrants the development of alternative strategies for the treatment of iron overload cardiomyopathies. Our results suggest that inhibition of Fe²⁺ uptake by Ca²⁺ channel blockers might be useful for the clinical management of iron overload disorders. Finally, our observations could also be of significance in other tissues, such as pancreas and pituitary glands, which also possess high L-type Ca²⁺ channel activity^{62 63} for hormone secretion, because iron overload is commonly associated with both diabetes and pituitary hormone dysfunction.^{4 5 7}

	Acknowledgments

 
This work was supported by the Heart and Stroke Foundation of Ontario and the Medical Research Council of Canada (to P.H.B.). A.D.W. was supported by a fellowship from the Center for Cardiovascular Research. P.H.B. is a Medical Research Council of Canada scholar.

Received September 9, 1998; accepted March 25, 1999.

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